Hypertension
Hypertension is a condition that occurs when the blood pressure inside the large arteries is too high. Hypertension is very common, affecting about 50 million people in the United States alone. It is more common as people grow older and is both more common and more serious in African Americans. Most cases of hypertension are of unknown etiology. It is known that the tendency to develop hypertension can be inherited. Environment also plays a very important role in hypertension. For example, hypertension may be avoided by keeping body weight under control, keeping physically fit, eating a healthy diet, limiting alcohol intake, and avoiding medications that might increase blood pressure. Other less common causes of hypertension include disorders of the kidneys or endocrine glands. Hypertension has been called “the silent killer” because it has no specific symptoms and yet can lead to death. People with untreated hypertension are much more likely to die from or be disabled by cardiovascular complications such as strokes, heart attacks, heart failure, heart rhythm irregularities, and kidney failure, than people who have normal blood pressure.
Current treatments for hypertension include lifestyle changes (diet, exercise, nonsmoking, etc.) as well as drug therapy. The major classes of medications currently used to treat hypertension include adrenergic neuron antagonists (which are peripherally acting), alpha adrenergic agonists (which are centrally acting), alpha adrenergic blockers, alpha & beta blockers, angiotensin II receptor blockers, angiotensin converting enzyme (ACE) inhibitors, beta adrenergic blockers, calcium channel blockers, Thiazide and related diuretics, and vasodilators (which act by direct relaxation of vascular smooth muscles).
A particularly serious hypertensive disorder is primary pulmonary hypertension, also known as idiopathic pulmonary hypertension. This is a condition in which the blood pressure in the pulmonary arteries is abnormally high in the absence of other diseases of the heart or lungs. The cause of primary pulmonary hypertension is unknown. Pulmonary hypertension develops in response to increased resistance to blood flow. Narrowing of the pulmonary arterioles occurs and the right side of the heart becomes enlarged due to the increased work of pumping blood against the resistance. Eventually, progressive heart failure develops. Currently, there is no known cure for primary pulmonary hypertension. Treatment is primarily directed towards controlling the symptoms, although some success has occurred with the use of vasodilators. Other medications used to treat the symptoms of primary pulmonary hypertension include diuretics and calcium channel blockers. Typically, as the disease progresses, oxygen if often required. In certain cases, a heart-lung transplant may be indicated for certain suitable candidates, although the availability of donor organs continues to be extremely limited. Unfortunately, primary pulmonary hypertension is a progressive disease, usually leading to congestive heart failure and respiratory failure.
Secondary pulmonary hypertension is a serious disorder that arises as a complication of other conditions such as, for example, scleroderma. Treatments are similar as those for primary pulmonary hypertension and, unfortunately, the prognosis is the same as well.
Chronic circulatory disorders
Ischemia is a condition in which the blood flow within an artery is limited to the point where the oxygen needs of the organ it serves cannot be met, thus resulting in hypoxia. Different types of ischemia have different names, depending on which organ in the body is deprived of oxygen. For example, reduced blood flow to the brain is called cerebral ischemia, which can lead to either a warning stroke called a transient ischemic attack (TIA) or possibly a full stroke. Cardiac ischemia is a situation in which the blood flow within a coronary artery is limited to the point where the oxygen needs of the heart muscle cannot be met. Cardiac ischemia can lead to myocardial infarction (heart attack). Other types of ischemia include hepatic ischemia and renal ischemia. Additionally, ischemia within the lower extremities (e.g., the legs) can cause muscle cramping when walking and in more severe cases can cause pain in the feet even at rest. In the most extreme cases gangrene can form. This occurs when local tissues are deprived of oxygen such that they die. Unfortunately, gangrene often leads to amputation of the involved limb. This type of ischemia is common in diabetics.
Minor episodes of cardiac ischemia tend to cause little long-term damage to the heart, but these episodes can sometimes cause serious problems. For example, they can cause arrhythmias, which can lead to either fainting or even cardiac arrest. However, severe or lengthy episodes of cardiac ischemia can trigger a heart attack. The collective effects of minor episodes of cardiac ischemia can potentially lead to a weakening of the heart muscle, a condition known as cardiomyopathy. Extreme cases of cardiomyopathy can require a heart transplant to save the individuals life.
Because cardiac ischemia occurs when the heart is not getting enough oxygen, one treatment may be to reduce the heart's need for oxygen. This treatment involves taking medications that slow heart rate, reduce blood pressure, and relax the blood vessels (vasodilation). The three main classes of drugs that accomplish this are called beta-blockers, calcium channel blockers, and nitrates. In addition, aspirin and other anti-platelets are very useful in decreasing blood clot formation, thus reducing the chance of blocked arteries triggering a heart attack. Exercise and/or stress management techniques may be helpful as well. More invasive techniques such as a balloon angioplasty or bypass surgery may be recommended if the less invasive treatments are unsuccessful.
Other, less serious, ischemic conditions exist as well. Buerger's disease, or thromboangiitis obliterans, is characterized by an inflammatory response in the arteries, veins, and nerves, which leads to a thickening of the blood vessel walls caused by infiltration of white blood cells. The cause of Buerger's disease is unknown, but because it occurs mostly in young men who smoke, it is thought to be a reaction to something in cigarettes or the result of a genetic or autoimmune disorder. The first symptoms are usually a bluish cast to a toe or finger and a feeling of coldness in the affected limb. As the blood vessels become blocked, intermittent claudication (a condition caused by diminished blood supply to the leg muscles as a result of the narrowing of arteries of the legs; marked by pain and lameness during mobility, but absence of pain when sedentary) and other symptoms similar to those of chronic obstructive arterial disease often appear. Ischemic ulcers and gangrene are common complications of progressive Buerger's disease. The most effective and important treatment is to smoking cessation; if this is done early in the disease before serious blood vessel or nerve damage has occurred, the symptoms usually improve markedly. If pain and circulatory problems persist, surgery to sever the sympathetic nerves that cause the small blood vessels to constrict may be performed.
Raynaud's phenomenon is characterized by spasms of the arteries in the fingers and toes, causing a lack of blood flow to the affected parts. There are two classifications of this disorder: primary, in which there is no evidence of other underlying disease; and secondary, in which the condition is complicated by other disorders, such as lupus erythematosus, rheumatoid arthritis, or scleroderma. Sometimes Raynaud's may accompany Buerger's disease. Excessive constriction of the vessels serving the fingers and toes causes spasms, which are usually triggered by cold, smoking cigarettes, or, less frequently, emotional factors. Raynaud's phenomenon may be an indication of an underlying disease. The symptoms include coldness, blueness, a tingling sensation, numbness, and sometimes pain in the digits. Corresponding fingers on both hands are usually affected. Primary Raynaud's may be uncomfortable or annoying, but rarely leads to serious problems such as chronic ulcers or gangrene. Many people with Raynaud's phenomenon get some relief by using a calcium-channel blocker such as nifedipine. Another medication called phenoxybenzamine may also help by limiting the constricting effects of adrenaline on the blood vessels. In some cases, the circulation may be impaired enough to cause sores or ulcers to form; in a small minority, these may progress to gangrene and amputation.
Acute Ischemias
Stroke syndrome is caused by a disorder of the blood vessels serving the brain, resulting in cerebral ischemia. Cerebral ischemia is also called stroke, cerebral vascular accident, and cerebrovascular accident. There are four neurological events associated with stroke: transient ischemic attack (TIA), reversible ischemic neurologic deficit (RIND), stroke in evolution (SIE), and completed stroke (CS). TIAs are temporary attacks that come on suddenly and last only a few minutes to not more than 24 hours; although they often are not recognized as such, they are a warning that a completed stroke can occur. RIND is an event similar to TIA except that the symptoms last for several days to a week; there is complete or nearly complete recovery. Like TIA, RIND is an indication that the person is at high risk for a completed stroke. A person with a stroke in evolution (SIE) experiences gradual weakness on one side of the body. The diagnosis of SIE is confirmed when the progressive changes are witnessed by the physician. The person with a completed stroke (CS) exhibits symptoms associated with severe cerebral ischemia resulting from an interrupted blood supply to the brain. Persons most at risk for any of the four types of stroke include those with hypertension, atherosclerosis, and heart disease and other cardiovascular disorders. Obese persons, heavy smokers, and those with diabetes mellitus are also at increased risk.
Medical and surgical preventive measures have significantly reduced the incidence of stroke in the United States. Medical preventive measures are aimed at eliminating or controlling atherosclerosis and other conditions that predispose a person to stroke. Effective control of hypertension and treatment of rheumatic and atherosclerotic heart disease have significantly reduced the incidence of stroke. Efforts to control diabetes mellitus, reduce cholesterol levels by diet and exercise, manage obesity, and encourage cessation of smoking are all examples of measures that have been successful in preventing stroke in significant numbers of people at risk. The choice of medical prevention and treatment is governed by the conditions that predispose the patient to having a stroke and, in the event one has already occurred, the potential of the individual patient to benefit from the treatment.
Other disorders associated with vascular dysfunction and insufficiency
Penile erectile dysfunction is a disorder characterized by an inability to achieve or maintain an erection and most commonly occurs when the penis is deprived of oxygen-rich blood. A number of conditions can deprive the penis of oxygen-rich blood. The primary cause of oxygen deprivation is ischemia—the blockage of blood vessels. The same conditions, such as unhealthy cholesterol levels, that cause blockage in the blood vessels leading to heart problems may also contribute to erectile dysfunction. As the plaque builds up, the arterial walls slowly constrict, reducing blood flow; this process, known as atherosclerosis, is the major contributor to the development of coronary heart disease. It may also play a role in the development of erectile dysfunction.
Nitric Oxide
The importance of endothelial nitric oxide (NO) generation in sustaining a tonic systemic vasodilatation is well established. Inhibiting NO production produces hypertension in animals and in humans and not surprisingly there has been considerable interest in establishing whether deficiencies of endothelial NO pathway activity are implicated in the etiology of hypertension.
Angiopoietins
An angiogenic factor, which was originally called TIE-2 ligand-1 (TL1) but is also referred to as angiopoietin-1 (Ang1), has been previously identified. Ang1 signals through the endothelial cell-specific receptor called the TIE-2 receptor and is essential for normal vascular development. By homology screening, an Ang1 relative has also been identified and is called TIE-2 ligand-2 (TL2) or angiopoietin-2 (Ang2). Ang2 is a naturally occurring context-specific antagonist for Ang1 and the TIE-2 receptor. For a description of the cloning and sequencing of TL1 (Ang1) and TL2 (Ang2) as well as for methods of making and uses thereof, reference is hereby made to PCT International Publication No. WO 96/11269 published Apr. 18, 1996 and PCT International Publication No. WO 96/31598 published Oct. 10, 1996 both in the name of Regeneron Pharmaceuticals, Inc.; and S. Davis, et al., Cell 87: 1161–1169 (1996) each of which is hereby incorporated by reference. In addition to the naturally occurring angiopoietins, several mutant molecules have been engineered that exhibit improved properties over the angiopoietins. For example, Ang1* is a mutant form of Ang1 that comprises the N-terminal domain of Ang2 fused to the coiled-coil domain and the fibrinogen domain of Ang1 and that has a Cys to Ser mutation at amino acid 245. Ang1* has been shown to be a potent agonist for the Tie-2 receptor (See U.S. Pat. No. 6,265,564, issued Jul. 24, 2001, to Davis, et al. which is hereby incorporated by reference).
In addition to the above-described angiopoietins, several related angiogenic factors have been identified. These have been designated Tie ligand-3 (TL3, Ang3) and Tie ligand-4 (TL4, Ang4). For descriptions of the structure and functional properties of these four related factors, reference is hereby made to the following publications, each of which is hereby incorporated by reference: U.S. Pat. No. 5,851,797, issued Dec. 22, 1998, in the name of Davis, et al.; PCT International Application No. PCT/US95/12935, filed Oct. 6, 1995, published on Apr. 18, 1996, with Publication No. WO 96/11269; PCT International Application No. PCT/US96/04806, filed Apr. 5, 1996, published on Oct. 10, 1996, with Publication No. WO96/31598; PCT International Application No. PCT/US97/10728, filed Jun. 19, 1997, published on Dec. 24, 1997 with Publication No. WO 97/48804. All PCT applications filed in the name of Regeneron Pharmaceuticals, Inc.
The angiopoietins can be structurally divided into three domains: an N-terminal region lacking in homology to any known structures; an alpha helical rich coil-coil segment similar to motifs found in many proteins that seem to promote multimerization; and a “fibrinogen-like domain” thus dubbed because it is distantly related to a domain first found in fibrinogen but now noted to be in many other proteins (Davis, S. et al., (1996) Cell 87:1161–1169). The fibrinogen-like domain represents the most conserved region of the angiopoietins, and recent studies indicate that it comprises the receptor-binding portion of an angiopoietin. In addition, all the information that determines whether an angiopoietin is an agonist or an antagonist appears to reside within the fibrinogen-like domain. For example, when chimeric molecules are made in which the fibrinogen-like domains of angiopoietin-1 and angiopoietin-2 are swapped, agonistic or antagonistic abilities track with the fibrinogen-like domains. The N-terminal and coil-coil regions appear to serve mainly to multimerize the fibrinogen-like domains, which apparently must be clustered to be active. In fact, the N-terminal and coil-coil regions can be substituted for by alternative motifs that allow clustering. Thus, the activities of Ang1 and Ang2 can be precisely mimicked by surrogates in which the fibrinogen-like domains (FD) of these factors are fused to the constant region of an antibody, resulting in FD-Fc fusions, which can then be clustered using secondary antibodies directed against the Fc. For a general description of the production and use of FD-Fc fusions, see International Publication Number WO 97/48804 published Dec. 24, 1997. Using these techniques, one of skill in the art would be able to similarly make FD-Fc fusions using the fibrinogen-like domain of an angiopoietin family member. One practical advantage of such surrogates is that native angiopoietins can be difficult to produce recombinantly, while the surrogates can be more easily produced.
As described supra, experiments with mutants of Ang1 and Ang2 have demonstrated that the fibrinogen domains (FD) are the receptor-binding domains, and that dimerized versions (dimerization occurs due to the interaction between the Fc components of adjacent molecules), for example Ang1-FD-Fc, can bind to the TIE2 receptor with much higher affinity than monomeric Ang1-FD. However, Ang1-FD-Fc is not able to induce phosphorylation (activate) the TIE2 receptor on endothelial cells unless it is further clustered with goat anti-human Fc antibodies (Jackson Immunoresearch). For this reason, mutant versions of Ang1-FD and Ang2-FD were designed that were intrinsically more highly clustered.
Two general types of nucleic acid molecules were constructed. The first type consisted of two tandem copies of Ang1-FD fused to an Fc tag, thus leading to a secreted polypeptide molecule that is dimeric with respect to the Fc tag but tetrameric with respect to Ang1-FD. Similarly, two tandem copies of Ang2-FD fused to an Fc tag constituted the Ang2 version of this type of construct. These molecules were designated Ang1-FD-FD-Fc and Ang2-FD-FD-Fc, respectively.
In the second type of nucleic acid molecule constructed, two copies of Ang1-FD were connected by an Fc tag bridging between them, thus creating the structure Ang1-FD-Fc-FD that is still dimeric with respect to the Fc, as well as tetrameric with respect to Ang1-FD. An Ang2 version was also constructed and these two molecules were designated Ang1-FD-Fc-FD and Ang2-FD-Fc-FD, respectively.
For either type of construct, similar properties were observed: unlike dimeric Ang1-FD-Fc, which fails to activate TIE2 on endothelial cells, both Ang1-FD-FD-Fc and Ang1-FD-Fc-FD could readily activate TIE2 on endothelial cells, with a potency comparable to that of the native ligand. Also, like native Ang-2, Ang2-FD-Fc-FD could behave as a context-specific antagonist of Ang1 activity with a potency that is comparable to that of native Ang2, and with much greater potency than the marginally antagonistic activity of the Ang2-FD-Fc dimer.
In accordance with the subject invention, Applicants describe herein methods of treating vascular diseases, in particular, hypertension and ischemia, by the administration of TIE2 receptor activating molecules. Such molecules include, but are not limited to, Ang1, Ang1*, Ang1-FD-Fc-FC and other suitable fragments and derivatives capable of activating TIE2.